Potential factors contributing to the increased risk for cognitive impairment (CI) in type 2 diabetes (T2D) include: (a) components of Alzheimer's disease (AD) pathology (plaques, tangles, synapse loss, neuronal loss); (b) atherosclerotic vasculopathy; (c) brain insulin resistance; (d) inflammation; (e) prior episodes of hypoglycemia; (f) other, as yet unknown factors. In the only report of this topic in which AD brain has been assessed directly, Talbot et al presented evidence in support of the hypothesis that insulin resistance is a consistent feature of all typical, sporadic AD. Project 2 focuses on the putative pathophysiological underpinnings between insulin resistance/T2D and CI. Investigators in Project 2 will use an induced pluripotent stem cell (iPSC) strategy to derive neurons, astrocytes, mixed brain cell cultures, and white adipocytes from various clinical populations defined in Project 1. The neurons, astrocytes, and mixed cultures will be used to study the cellular phenotypes and insulin sensitivities of central nervous system (CNS) cells, while the adipocytes will be used as exemplars of peripheral insulin-sensitive cells. We will assess quantitatively the insulin sensitivities in CNS and peripheral cells derived from iPSCs from various clinical populations defined in Project 1. In order to establish the insulin sensitivity of the iPSC-derived neuron, we will study classical insulin signaling pathways in all cell types as assessed through the phosphorylation state of downstream signaling molecules. Importantly, as a physiological readout for insulin action, we will study neurons by electrophysiology and calcium imaging, while adipocytes will be characterized through the assessment of the ability of insulin to increase glucose uptake and to suppress lipolysis. To ascertain the dependence of these responses of insulin signaling through the insulin receptor, we will employ both pharmacological or molecular approaches, the latter via an antisense-mediated knockdown of the insulin receptor or the expression of a dominant-negative mutant version of the insulin-like growth factor (IGF)-1 receptor that heterodimerizes with the insulin receptor and blocks its function. These studies will establish whether insulin resistance is a feature of AD in peripheral and/or brain cells. Additional studies will provide a direct assessment for the possible participation of insulin resistance in the generation of structural pathology causing or predisposing to CI. With regard to pathology, we will measure insulin-stimulated A? secretion, and insulin-modulated tau phosphorylation in brain cells derived from the clinical populations defined in Project 1. Overall, the data derived from this project will test the hypothesis that insulin resistance is a consistent feature of the sporadic AD phenotype.